Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish

Summary

Zebrafish embryos/larvae develop externally and are optically transparent. The bioaccumulation of microplastics in fish at early life stages is readily assessed with fluorescently labeled microbeads.

Abstract

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The bioaccumulation of microplastics plays a key role in their toxic effects; however, as a particulate, their bioaccumulations are different from many other pollutants. Described here is a feasible method to visually determine the accumulation and distribution of microplastics in zebrafish embryos or larvae using fluorescent microplastics. Embryos are exposed to different concentrations (0.1, 1, and 10 mg/L) of fluorescent microplastics with a diameter of 500 nm for 120 h. It is shown in the results that microplastics can bioaccumulate in zebrafish embryos/larvae in a concentration-dependent manner. Before hatching, strong fluorescence is found around the embryonic chorion; while in zebrafish larvae, the yolk sac, pericardium, and gastrointestinal tract are the main accumulated sites of microplastics. The results demonstrate the uptake and internalization of microplastics in zebrafish at early life stages, which will provide basis for better understanding the impact of microplastics on aquatic animals.

Introduction

Since first synthesized in the 1900s, plastics are widely used in various fields, resulting in rapid growth of global production¹. In 2018, approximately 360 million tons of plastics were produced worldwide². The plastics in the natural environment will degrade to fine particles due to chemical, physical or biological processes³. Generally, fine plastic particles <5 mm in size are defined as microplastics⁴. Microplastics are also engineered for specific applications, such as microbeads from cosmetic products⁵. As near-permanent contaminants, microplastics are accumulated in the environment, and have attracted increasing attention from scientists, policymakers and the public^1,6. Previous studies documented that microplastics could cause adverse effects in fish, such as gastrointestinal damage⁷, neurotoxicity⁸, endocrine disruption⁹, oxidative stress¹⁰ and DNA damage¹¹. However, the toxicity of microplastics has not been fully revealed so far^12,13.

Zebrafish embryos offer a lot of experimental advantages, including small size, external fertilization, optical transparency and large clutches, and is considered as an ideal model organism for in vivo studying the effects of pollutants on fish at early life stages. In addition, only limited amounts of test substances are needed for the evaluation of biological responses. Here, zebrafish embryos are exposed to different concentrations of microplastics (0.1, 1, 10 mg/L) for 5 days, and the bioaccumulation and distribution of microplastics in zebrafish embryos/larvae are evaluated. This result will advance our understanding about the toxicity of microplastics to fish, and the method described here can potentially be generalized to determine the accumulation and distribution of other types of fluorescent materials in the early life stages of zebrafish.

Protocol

Adult zebrafish are originated from the China Zebrafish Resource Center (Wuhan, China). The experiments were conducted in compliance with the national guide "Laboratory Animal Guideline for Ethical Review of Animal Welfare (GB/T35892-2018).

1. Embryo collection

1. Maintain fish in 20 L glass tanks with recirculating charcoal-filtered tap water system (pH 7.0 ± 0.2) at a constant temperature (28 ± 0.5 °C) on a photoperiod of 14:10 h light: dark.
2. Feed fish twice daily with Artemia nauplii. It is recommended that the food is given at max. 3% fish weight per day and should be eaten within 5 min every time¹⁴.
3. Transfer well-developed adult zebrafish (with body length of 3-4 cm) into the spawning tank at a ratio of one male to two females the night before the breeding.
   NOTE: The following morning, the fish start to spawn after the onset of the light cycle.
4. Collect eggs using a Pasteur pipette. Rinse with 10% Hank's solution several times, and then check for fertilization using a microscope. Fertilized eggs undergo the cleavage period after approximately 2 h post fertilization (hpf) and can be clearly identified¹⁵.
5. Incubate the fertilized embryos in a 500 mL beaker containing 200 mL of 10% Hank's solution with 1% methylene blue for disinfection at 28 °C. Do not exceed a loading rate of 1 embryo/2 mL solution.
   NOTE: 10% Hank's solution is made up of 137 mM NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 1.3 mM CaCl₂, 1.0 mM MgSO₄ and 4.2 mM NaHCO₃.

2. Preparation of microplastic suspensions

1. Sonicate the stock solution of green fluorescently labeled polystyrene beads (10 mg/mL) with nominal diameter of 500 nm (excitation/emission: 460/500 nm) for 10 minutes.
2. Dilute the stock solution with 10% Hank's solution to produce the desired exposure solutions (0.1, 1, and 10 mg/L).
3. Always prepare the exposure solutions of microplastics before exposure.
   ​NOTE: Caution should be taken when assessing the toxic effects of microplastics, because the presence of preservatives, such as sodium azide, in the commercial particle formulations, can be toxic to different organisms ¹⁶. Therefore, these additives should be removed or accounted for in the controls before conducting toxicity experiment.

3. Microplastic exposure

1. Randomly select 6 newly fertilized embryos (4 hpf), and then transfer into each well of 6-well plate containing 5 mL of microplastic solutions with different concentrations. Include the control groups containing 10% Hank's solution.
   1. Use triplicate wells (with a total of 18 embryos) for each treatment.
2. Incubate the embryos under the same light: dark cycle and temperature as adults (see 1.2) and observe every 12 hours. Remove the dead immediately.
3. Renew the microplastic solutions 90% every 24 h. During the exposure period, the fish are not fed.
   NOTE: Generally, the hatching of embryo begins at 48 hpf and completes at about 72 hpf.

4. Assessment of microplastic distribution

1. At 24, 48, 72, 96, and 120 h post fertilization, randomly select the embryos/larvae (one from each of the three replicates) and rinse with 10% Hank's solution.
2. Transfer the larvae into a Petri dish and expose to 0.016% tricaine for anesthesia.
   1. Prepare the stock solution of tricaine: 4 mg of tricaine powder is dissolved in 100 mL of double distilled water, and adjust the pH to 7.0 with Tris-HCl (pH 9.0). Store the stock solution in the freezer.
   2. Prepare the working solution. Dilute the stock solution to the desired concentration (0.016%) with 10% Hank's solution at room temperature¹⁴.
3. Arrange the embryos/larvae and prepare for observation.
4. Observe the fish with a fluorescence microscope and image with imaging software.
5. Quantify the fluorescence intensity in fish with ImageJ.

Results

The distribution and accumulation of fluorescent microplastics are shown in Figure 1 and Table 1. No visible fluorescence is observed in the unexposed group (control). However, an accumulation of fluorescence is found surrounding the chorion after exposure to different concentrations of microplastics (24 hpf). Green fluorescence is also detected in larvae, and the fluorescence levels appear to increase in a concentration- and time-dependent manner. The yolk sac, pericardium,...

Discussion

According to the guideline on the protection of animals used for scientific purposes, such as EU Directive 2010/63/EU, animal ethics permission is not mandatory for an experiment with early life-stages of zebrafish until the stage of being capable of independent feeding (5 days post fertilization)¹⁷. However, best welfare practice is important for optimizing the use of zebrafish, and, for example, the humane methods of anesthesia and euthanasia should be of concern. Ethyl 3-aminobenzoate methanesu...

Materials

Name	Company	Catalog Number	Comments
Fluorescent microscope	Nikon, Japan	Eclipse Ti-S
Green fluorescently labeled polystyrene beads	Phosphorex, USA	2103A
Tricaine	Sigma-Aldrich, USA	A5040